Relationship of the bone phenotype of the Klotho mutant mouse model of accelerated aging to changes in skeletal architecture that occur with chronological aging.

Introduction: Due to the relatively long life span of rodent models, in order to expediate the identification of novel therapeutics of age related diseases, mouse models of accelerated aging have been developed. In this study we examined skeletal changes in the male and female Klotho mutant (kl/kl) mice and in male and female chronically aged mice to determine whether the accelerated aging bone phenotype of the kl/kl mouse reflects changes in skeletal architecture that occur with chronological aging. Methods: 2, 6 and 20-23 month old C57BL/6 mice were obtained from the National Institute of Aging aged rodent colony and wildtype and kl/kl mice were generated as previously described by M. Kuro-o. Microcomputed tomography analysis was performed ex vivo to examine trabecular and cortical parameters from the proximal metaphyseal and mid-diaphyseal areas, respectively. Serum calcium and phosphate were analyzed using a colorimetric assay. The expression of duodenal Trpv6, which codes for TRPV6, a vitamin D regulated epithelial calcium channel whose expression reflects intestinal calcium absorptive efficiency, was analyzed by quantitative real-time PCR. Results and discussion: Trabecular bone volume (BV/TV) and trabecular number decreased continuously with age in males and females. In contrast to aging mice, an increase in trabecular bone volume and trabecular number was observed in both male and female kl/kl mice. Cortical thickness decreased with advancing age and also decreased in male and female kl/kl mice. Serum calcium and phosphate levels were significantly increased in kl/kl mice but did not change with age. Aging resulted in a decline in Trpv6 expression. In the kl/kl mice duodenal Trpv6 was significantly increased. Our findings reflect differences in bone architecture as well as differences in calcium and phosphate homeostasis and expression of Trpv6 between the kl/kl mutant mouse model of accelerated aging and chronological aging. Although the Klotho deficient mouse has provided a new understanding of the regulation of mineral homeostasis and bone metabolism, our findings suggest that changes in bone architecture in the kl/kl mouse reflect in part systemic disturbances that differ from pathophysiological changes that occur with age including dysregulation of calcium homeostasis that contributes to age related bone loss.

Role of Coactivator Associated Arginine Methyltransferase 1 (CARM1) in the Regulation of the Biological Function of 1,25-Dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, activates the nuclear vitamin D receptor (VDR) to mediate the transcription of target genes involved in calcium homeostasis as well as in non-classical 1,25(OH)2D3 actions. In this study, CARM1, an arginine methyltransferase, was found to mediate coactivator synergy in the presence of GRIP1 (a primary coactivator) and to cooperate with G9a, a lysine methyltransferase, in 1,25(OH)2D3 induced transcription of Cyp24a1 (the gene involved in the metabolic inactivation of 1,25(OH)2D3). In mouse proximal renal tubule (MPCT) cells and in mouse kidney, chromatin immunoprecipitation analysis demonstrated that dimethylation of histone H3 at arginine 17, which is mediated by CARM1, occurs at Cyp24a1 vitamin D response elements in a 1,25(OH)2D3 dependent manner. Treatment with TBBD, an inhibitor of CARM1, repressed 1,25(OH)2D3 induced Cyp24a1 expression in MPCT cells, further suggesting that CARM1 is a significant coactivator of 1,25(OH)2D3 induction of renal Cyp24a1 expression. CARM1 was found to act as a repressor of second messenger-mediated induction of the transcription of CYP27B1 (involved in the synthesis of 1,25(OH)2D3), supporting the role of CARM1 as a dual function coregulator. Our findings indicate a key role for CARM1 in the regulation of the biological function of 1,25(OH)2D3.

Intestinal Vitamin D Receptor Is Dispensable for Maintaining Adult Bone Mass in Mice With Adequate Calcium Intake.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)-mediated intestinal calcium (Ca) absorption supplies Ca for proper bone mineralization during growth. We tested whether vitamin D receptor (VDR)-mediated 1,25(OH)2D3 signaling is critical for adult Ca absorption and bone by using mice with inducible Vdr gene knockout in the whole intestine (villin-CreERT2+/- × Vdrf/f, WIK) or in the large intestine (Cdx2-CreERT2+/- ×Vdrf/f, LIK). At 4-month-old, Vdr alleles were recombined (0.05 mg tamoxifen/g BW, intraperitoneally [i.p.], 5 days) and mice were fed diets with either 0.5% (adequate) or 0.2% (low) Ca. Ca absorption was examined after 2 weeks while serum 1,25(OH)2D3, bone mass, and bone microarchitecture were examined after 16 weeks. Intestinal and renal gene expression was measured at both time points (n = 12/genotype/diet/time point). On the 0.5% Ca diet, all phenotypes in WIK and LIK mice were similar to the controls. Control mice adapted to the 0.2% low-Ca diet by increasing renal Cyp27b1 mRNA (3-fold), serum 1,25(OH)2D3 level (1.9-fold), and Ca absorption in the duodenum (Dd, + 131%) and proximal colon (PCo, + 28.9%), which prevented bone loss. In WIK mice, low-Ca diet increased serum 1,25(OH)2D3 (4.4-fold) but Ca absorption remained unaltered in the Dd and PCo. Consequently, significant bone loss occurred in WIK mice (e.g., cortical thickness, Ct.Th, -33.7%). LIK mice adapted to the low-Ca diet in the Dd but not the PCo, and the effect on bone phenotypes was milder (e.g., Ct.Th, -13.1%). Our data suggest intestinal VDR in adult mice prevents bone loss under low Ca intake but is dispensable under adequate calcium intake.

Highlights from the 24th workshop on vitamin D in Austin, September 2022.

The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.

Genomic analysis of 1,25-dihydroxyvitamin D3 action in mouse intestine reveals compartment and segment-specific gene regulatory effects.

1,25-dihydroxyvitamin D (VD) regulates intestinal calcium absorption in the small intestine (SI) and also reduces risk of colonic inflammation and cancer. However, the intestine compartment-specific target genes of VD signaling are unknown. Here, we examined VD action across three functional compartments of the intestine using RNA-seq to measure VD-induced changes in gene expression and Chromatin Immunoprecipitation with next generation sequencing to measure vitamin D receptor (VDR) genomic binding. We found that VD regulated the expression of 55 shared transcripts in the SI crypt, SI villi, and in the colon, including Cyp24a1, S100g, Trpv6, and Slc30a10. Other VD-regulated transcripts were unique to the SI crypt (162 up, 210 down), villi (199 up, 63 down), or colon (102 up, 28 down), but this did not correlate with mRNA levels of the VDR. Furthermore, bioinformatic analysis identified unique VD-regulated biological functions in each compartment. VDR-binding sites were found in 70% of upregulated genes from the colon and SI villi but were less common in upregulated genes from the SI crypt and among downregulated genes, suggesting some transcript-level VD effects are likely indirect. Consistent with this, we show that VD regulated the expression of other transcription factors and their downstream targets. Finally, we demonstrate that compartment-specific VD-mediated gene expression was associated with compartment-specific VDR-binding sites (<30% of targets) and enrichment of intestinal transcription factor-binding motifs within VDR-binding peaks. Taken together, our data reveal unique spatial patterns of VD action in the intestine and suggest novel mechanisms that could account for compartment-specific functions of this hormone.

Regulatory domains controlling high intestinal vitamin D receptor gene expression are conserved in mouse and human.

Vitamin D receptor (VDR) levels are highest in the intestine where it mediates 1,25 dihydroxyvitamin D-induced gene expression. However, the mechanisms controlling high intestinal VDR gene expression are unknown. Here, we used Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) to identify the regulatory sites controlling intestine-specific Vdr gene expression in the small intestine (villi and crypts) and colon of developing, adult, and aged mice. We identified 17 ATAC peaks in a 125 kb region from intron 3 to -55.8 kb from exon 1 of the Vdr gene. Interestingly, many of these peaks were missing/reduced in the developing intestine. Chromatin ImmunoPrecipitation-Sequencing (ChIP-Seq) peaks for intestinal transcription factors (TFs) were present within the ATAC peaks and at HiChIP looping attachments that connected the ATAC/TF ChIP peaks to the transcription start site and CCCTF-binding factor sites at the borders of the Vdr gene regulatory domain. Intestine-specific regulatory sites were identified by comparing ATAC peaks to DNAse-Seq data from other tissues that revealed tissue-specific, evolutionary conserved, and species-specific peaks. Bioinformatics analysis of human DNAse-Seq peaks revealed polymorphisms that disrupt TF-binding sites. Our analysis shows that mouse intestinal Vdr gene regulation requires a complex interaction of multiple distal regulatory regions and is controlled by a combination of intestinal TFs. These intestinal regulatory sites are well conserved in humans suggesting that they may be key components of VDR regulation in both mouse and human intestines.

Vitamin D: A Critical Regulator of Intestinal Physiology.

Calcium is required for the functioning of numerous biological processes and is essential for skeletal health. The major source of new calcium is from the diet. The central role of vitamin D in the maintenance of calcium homeostasis is to increase the absorption of ingested calcium from the intestine. The critical importance of vitamin D in this process is noted in the causal link between vitamin D deficiency and rickets, as well as in studies using genetically modified mice including mice deficient in the vitamin D receptor (Vdr null mice) or in the cytochrome P-450 enzyme, 25-hydroxyvitamin D3-1α- hydroxylase (CYP27B1) that converts 25-hydroxyvitamin D3 to the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (Cyp27b1 null mice). When these mice are fed diets with high calcium and lactose, rickets is prevented. The studies using mouse models provide supporting evidence indicating that the major physiological function of 1,25(OH)2D3/VDR is intestinal calcium absorption. This review summarizes what is known about mechanisms involved in vitamin D-regulated intestinal calcium absorption. Recent studies suggest that vitamin D does not affect a single entity, but that a complex network of calcium-regulating components is involved in the process of 1,25(OH)2D3-mediated active intestinal calcium absorption. In addition, numerous 1,25(OH)2D3 actions in the intestine have been described independent of calcium absorption. Although the translatability to humans requires further definition, an overview is presented that provides compelling evidence from the laboratory of 1,25(OH)2D3 intestinal effects, which include the regulation of adhesion molecules to enhance barrier function, the regulation of intestinal stem cell function, cellular homeostasis of other divalent cations, the regulation of drug metabolizing enzymes, and anti-inflammatory effects. © 2021 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Drivers of transcriptional variance in human intestinal epithelial organoids.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

1,25-Dihydroxyvitamin D3 and dietary vitamin D reduce inflammation in mice lacking intestinal epithelial cell Rab11a.

A number of studies have examined the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) on intestinal inflammation driven by immune cells, while little information is currently available about its impact on inflammation caused by intestinal epithelial cell (IEC) defects. Mice lacking IEC-specific Rab11a a recycling endosome small GTPase resulted in increased epithelial cell production of inflammatory cytokines, notably IL-6 and early onset of enteritis. To determine whether vitamin D supplementation may benefit hosts with epithelial cell-originated mucosal inflammation, we evaluated in vivo effects of injected 1,25(OH)2 D3 or dietary supplement of a high dose of vitamin D on the gut phenotypes of IEC-specific Rab11a knockout mice (Rab11aΔIEC ). 1,25(OH)2 D3 administered at 25 ng, two doses per mouse, by intraperitoneal injection, reduced inflammatory cytokine production in knockout mice compared to vehicle-injected mice. Remarkably, feeding mice with dietary vitamin D supplementation at 20,000 IU/kg spanning fetal and postnatal developmental stages led to improved bodyweights, reduced immune cell infiltration, and decreased inflammatory cytokines. We found that these vitamin D effects were accompanied by decreased NF-κB (p65) in the knockout intestinal epithelia, reduced tissue-resident macrophages, and partial restoration of epithelial morphology. Our study suggests that dietary vitamin D supplementation may prevent and limit intestinal inflammation in hosts with high susceptibility to chronic inflammation.

Analysis of 1,25-Dihydroxyvitamin D3 Genomic Action Reveals Calcium-Regulating and Calcium-Independent Effects in Mouse Intestine and Human Enteroids.

Although vitamin D is critical for the function of the intestine, most studies have focused on the duodenum. We show that transgenic expression of the vitamin D receptor (VDR) only in the distal intestine of VDR null mice (KO/TG mice) results in the normalization of serum calcium and rescue of rickets. Although it had been suggested that calcium transport in the distal intestine involves a paracellular process, we found that the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated genes in the proximal intestine associated with active calcium transport (Trpv6, S100g, and Atp2b1) are also induced by 1,25(OH)2D3 in the distal intestine of KO/TG mice. In addition, Slc30a10, encoding a manganese efflux transporter, was one of the genes most induced by 1,25(OH)2D3 in both proximal and distal intestine. Both villus and crypt were found to express Vdr and VDR target genes. RNA sequence (RNA-seq) analysis of human enteroids indicated that the effects of 1,25(OH)2D3 observed in mice are conserved in humans. Using Slc30a10-/- mice, a loss of cortical bone and a marked decrease in S100g and Trpv6 in the intestine was observed. Our findings suggest an interrelationship between vitamin D and intestinal Mn efflux and indicate the importance of distal intestinal segments to vitamin D action.

New aspects of vitamin D metabolism and action - addressing the skin as source and target.

Vitamin D has a key role in stimulating calcium absorption from the gut and promoting skeletal health, as well as many other important physiological functions. Vitamin D is produced in the skin. It is subsequently metabolized to its hormonally active form, 1,25-dihydroxyvitamin D (1,25(OH)2D), by the 1-hydroxylase and catabolized by the 24-hydroxylase. In this Review, we pay special attention to the effect of mutations in these enzymes and their clinical manifestations. We then discuss the role of vitamin D binding protein in transporting vitamin D and its metabolites from their source to their targets, the free hormone hypothesis for cell entry and HSP70 for intracellular transport. This is followed by discussion of the vitamin D receptor (VDR) that mediates the cellular actions of 1,25(OH)2D. Cell-specific recruitment of co-regulatory complexes by liganded VDR leads to changes in gene expression that result in distinct physiological actions by 1,25(OH)2D, which are disrupted by mutations in the VDR. We then discuss the epidermis and hair follicle, to provide a non-skeletal example of a tissue that expresses VDR that not only makes vitamin D but also can metabolize it to its hormonally active form. This enables vitamin D to regulate epidermal differentiation and hair follicle cycling and, in so doing, to promote barrier function, wound healing and hair growth, while limiting cancer development.

Vitamin D and Bone.

Vitamin D is a principal factor required for mineral and skeletal homeostasis. Vitamin D deficiency during development causes rickets and in adults can result in osteomalacia and increased risk of fracture. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the hormonally active form of vitamin D, is responsible for the biological actions of vitamin D which are mediated by the vitamin D receptor (VDR). Mutations in the VDR result in early-onset rickets and low calcium and phosphate, indicating the essential role of 1,25(OH)2D3/VDR signaling in the regulation of mineral homeostasis and skeletal health. This chapter summarizes our current understanding of the production of the vitamin D endocrine hormone, 1,25(OH)2D3, and the actions of 1,25(OH)2D3 which result in the maintenance of skeletal homeostasis. The primary role of 1,25(OH)2D3 is to increase calcium absorption from the intestine and thus to increase the availability of calcium for bone mineralization. Specific actions of 1,25(OH)2D3 on the intestine, kidney, and bone needed to maintain calcium homeostasis are summarized, and the impact of vitamin D status on bone health is discussed.

Vitamin D and the intestine: Review and update.

The central role of vitamin D in calcium homeostasis is to increase calcium absorption from the intestine. This article describes the early work that served as the foundation for the initial model of vitamin D mediated calcium absorption. In addition, other research related to the role of vitamin D in the intestine, including those which have challenged the traditional model and the crucial role of specific calcium transport proteins, are reviewed. More recent work identifying novel targets of 1,25(OH)2D3 action in the intestine and highlighting the importance of 1,25(OH)2D3 action across the proximal/distal and crypt/villus axes in the intestine is summarized.

New developments in our understanding of vitamin metabolism, action and treatment.

Although vitamin D has been reported to have pleiotropic effects including effects on the immune system and on cancer progression, the principal action of vitamin D is the maintenance of calcium and phosphate homeostasis. The importance of vitamin D in this process is emphasized by the consequences of vitamin D deficiency which includes rickets in children and osteomalacia in adults. Vitamin D deficiency has also been reported to increase the risk of falls and osteoporotic fractures. Although vitamin D fortification of foods (including dairy products) has contributed to a marked decrease in rickets in the Western world, vitamin D deficiency in children and adults is still prevalent world-wide. This review summarizes new developments in our understanding of vitamin D endocrine system and addresses clinical syndromes related to abnormalities in vitamin D metabolism and action. In addition, the current understanding of the evaluation of vitamin D deficiency and sufficiency and recommendations for achieving vitamin D sufficiency are discussed.

PU.1 and epigenetic signals modulate 1,25-dihydroxyvitamin D3 and C/EBPα regulation of the human cathelicidin antimicrobial peptide gene in lung epithelial cells.

LL-37, the only known human cathelicidin which is encoded by the human antimicrobial peptide (CAMP) gene, plays a critical role in protection against bacterial infection. We previously demonstrated that cathelicidin is induced by 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) in human airway epithelial cells with a resultant increase in bactericidal activity. In this study we identify key factors that co-operate with 1,25(OH) 2 D 3 in the regulation of CAMP. Our results show for the first time that PU.1, the myeloid transcription factor (which has also been identified in lung epithelial cells), co-operates with the vitamin D receptor and CCAAT/enhancer binding protein α (CEBPα) to enhance the induction of CAMP in lung epithelial cells. Our findings also indicate that enhancement of 1,25(OH) 2 D 3 regulation of CAMP by histone deacetylase inhibitors involves co-operation between acetylation and chromatin remodeling through Brahma-related gene 1 (BRG1; a component of the SWItch/sucrose nonfermentable [SWI/SNF] complex). BRG1 can be an activator or repressor depending on BRG1-associated factors. Protein arginine methyltransferase 5 (PRMT5), a methlytransferase which interacts with BRG1, represses 1,25(OH) 2 D 3 induced CAMP in part through dimethylation of H4R3. Our findings identify key mediators involved in the regulation of the CAMP gene in lung epithelial cells and suggest new approaches for therapeutic manipulation of gene expression to increase the antibacterial capability of the airway.

Intestinal Regulation of Calcium: Vitamin D and Bone Physiology.

The principal function of vitamin D in the maintenance of calcium homeostasis is to increase intestinal calcium absorption. This conclusion was made from studies in vitamin D receptor (VDR) null mice which showed that rickets and osteomalacia were prevented when VDR null mice were fed a rescue diet that included high calcium, indicating that the skeletal abnormalities of the VDR null mice are primarily the result of impaired intestinal calcium absorption. Although vitamin D is critical for controlling intestinal calcium absorption, the mechanisms involved have remained incomplete. This chapter reviews studies, including studies in genetically modified mice, that have provided new insight and have challenged the traditional model of VDR-mediated calcium absorption.

In search of regulatory circuits that control the biological activity of vitamin D.

Although the cytochrome P450 CYP27B1 plays a critical role in vitamin D biology, the molecular mechanisms involved in regulation of CYP27B1 have remained undefined. A new study has identified a kidney-specific control module distal to the Cyp27b1 gene that mediates the basal activity and hormonal regulation of Cyp27b1 This work provides a novel mechanism indicating differential regulation of Cyp27b1 in renal and non-renal cells and has implications for vitamin D biology in multiple sclerosis and perhaps other autoimmune diseases as well.

Biology and Mechanisms of Action of the Vitamin D Hormone.

The central role of hormonal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is to regulate calcium and phosphorus homeostasis via actions in intestine, kidney, and bone. These and other actions in many cell types not involved in mineral metabolism are mediated by the vitamin D receptor. Recent studies using genome-wide scale techniques have extended fundamental ideas regarding vitamin D-mediated control of gene expression while simultaneously revealing a series of new concepts. This article summarizes the current view of the biological actions of the vitamin D hormone and focuses on new concepts that drive the understanding of the mechanisms through which vitamin D operates.